Among biochemists and chemical biologists, there is a continuous quest for chemical reactions that can potentially be carried out in biological systems. Such chemical reactions may have tremendous applications in various branches of life sciences. However, extremely few reactions can meet the strict requirements of complex biological systems viz., aqueous medium, near neutral pH, ambient temperature, no metal catalysis, no toxic by-product and excellent selectivity for the target functional group.
Proteins and the environment surrounding them are one such complex system, where conducting targeted chemical reactions in a bio-compatible way is still a challenge. Various sites in the protein structure have been targeted for this purpose, among which thiol group of the cysteine residue is one of the most common owing mainly to its high nucleophilicity and relatively low natural abundance (see Chalker, J. M. et al., Chemistry—An Asian Journal 4, 630-640, doi:10.1002/asia.200800427 (2009) and Fodje, M. N. et al., Protein Engineering 15, 353-358, doi:10.1093/protein/15.5.353 (2002)). However, a versatile reaction for proteins that meets all of the above mentioned requirements still remains elusive.
The most common methodologies, their drawbacks and challenges can be grouped as follows:                (a) classical reagents, such as maleimides, acrylamides and α-halo carbonyl compounds suffer mainly from lack of selectivity, their own stability and toxic by-products;        (b) metal catalyzed reactions that modify the cysteine residue show excellent selectivity (e.g. cross metathesis of allyl sulphides using Ruthenium catalyst by the group of B. G. Davis (Lin, Y. A., et al., Journal of the American Chemical Society 130, 9642-9643, doi:10.1021/ja8026168 (2008)) and thiol-allene coupling using gold catalyst by the group of C. M. Che (On-Yee Chan, A. et al., Chemical Communications 49, 1428-1430, doi:10.1039/C2CC38214H (2013)). However, the necessity to use metal complexes renders these reactions unfit for many biological applications;        (c) the use of electron deficient alkynes (Shiu, H.-Y. et al., Chemistry—A European Journal 15, 3839-3850, doi:10.1002/chem.200800669 (2009)), or bromomaleimides (Tedaldi, L. M., et al., Chemical Communications, 6583-6585, doi:10.1039/B915136B (2009) and Smith, M. E. B. et al., Journal of the American Chemical Society 132, 1960-1965, doi:10.1021/ja908610s (2010)) and dithiomaleimide derivatives (Robin, M. P. et al., Journal of the American Chemical Society 135, 2875-2878, doi:10.1021/ja3105494 (2013)). These reactions show excellent selectivity and have the advantage of being reversible under the presence of excess thiol. However, the reversibility poses a limitation when an in-vivo application is required because of the presence of a large excess of glutathione in the intracellular environment.        
Two groups (Ovaa and Mootz) have recently demonstrated simultaneously and independently that terminal alkynes (in modified ubiquitin) can be used to inhibit cysteine proteases irreversibly through in-situ thiol-alkyne coupling (Ekkebus, R. et al., Journal of the American Chemical Society 135, 2867-2870, doi:10.1021/ja309802n (2013) and Sommer, S., et al., Bioorganic & Medicinal Chemistry 21, 2511-2517, (2013)), which is quite impressive and surprising as alkynes have largely been considered inert under biological conditions. However, this coupling is not versatile as activation of an alkyne by a positively charged protein pocket (an oxoanion hole) has been proposed to be necessary for this unexpected reactivity (Arkona, C. et al., Angewandte Chemie International Edition, doi:10.1002/anie.201303544 (2013)).
With the long term goal to understand and regulate protein functions through their chemical modification, our group has previously reported modification of the N-terminus of peptides and proteins using Mukaiyama aldol condensation (Alam, J., et al., Journal of the American Chemical Society 132, 9546-9548, doi:10.1021/ja102733a (2010)).
However, there remains an urgent need to find a promising orthogonal handle and related labelling strategies that can selectively and irreversibly bind with cysteine, without the requirement for special local conditions (e.g. a positively charged protein pocket).